For many diseases, effective diagnosis and prognosis depends on the ability to quantitatively measure protein biomarkers present at low concentrations in clinical samples. For example, altered serum concentrations of cytokines, such as platelet-derived growth factor (PDGF) can point to tumor angiogenesis, while virus-related proteins, like hemagglutinin can indicate the extent of viral pathogen infection. Although blood serum is a rich source of diagnostic information, the analytical challenge in utilizing serum biomarkers arises from the fact that it contains thousands of proteins whose concentrations range over 12 orders of magnitude. Albumin, for example, constitutes approximately half of the serum proteins and is present at 30-50 mg/mL (0.5-0.8 mM), while many important biomarkers exist at concentrations at 1 pg/mL (10-100 fM). Due to this wide dynamic range, specific and quantitative detection of protein biomarkers in a single assay has been particularly difficult. Therefore, an innovative protein detection technologies with the following capabilities are critically needed: I) Integrated sample preparation - the sensor should have the capability to directly process clinical samples (i.e. whole blood or blood serum), II) the sensor should provide quantitative results of multiple markers that provides actionable diagnostic value, III) the sensor must have high sensitivity (femtomolar detection limits) and broad dynamic range, IV) the detection system must produce reproducible results and have low rates of false positives and negatives, V) the sensor should have a short total assay time and most importantly, VI) the total cost of the diagnostic information must be low. In order to address these challenges, a number of recent immunological methods such as ImmunoPCR, Proximity Ligation, and the Bio-Barcode assays have been developed to achieve detection performances beyond the capabilities of the enzyme-linked immunosorbent assay (ELISA), which as been the gold-standard for over three decades. However, such assays suffer from the fact that the reaction chemistry is hindered by the background serum proteins and other contaminants in the clinical samples. As a solution to this problem, we propose to integrate sample preparation, amplification and detection in a single disposable system such that we effectively transform a homogenous binding assay into a heterogeneous detection system. We propose to develop the the Micro-Magnetic Separation - Quantitative Polymerase Chain Reaction (MMS-QPCR) system wherein we integrate chip-based, high-gradient micro-magnetic separation with aptamer-based quantitative PCR to achieve quantitative, multiplexed protein detection with femtomolar detection sensitivities directly from undiluted serum. The project will be organized in three sections: first, we will design multiple sets of antibody - aptamer pairs to capture and label the target proteins with high affinity and specificity. Second, we will develop the micro-magnetic separation (MMS) chip to purify the target proteins from background serum for downstream detection. The use of magnetic particles will significantly improve the reaction kinetics, reduce incubation times, and eliminate extensive washing steps. The MMS chip will be directly interfaced as a front-end to a QPCR detection system. Thirdly, using the primer sequences imbedded in the aptamers, we will perform QPCR to achieve quantitative, multiplexed detection of target proteins at femtomolar concentrations. As a model system we will demonstrate simultaneous detection of three cancer markers, platelet derived growth factor (PDGF), hepatocyte growth factor (HGF), and thyroid transcription factor (TTF1), at femtomolar concentration levels directly from serum. From the preliminary results, we expect the limit of detection to be ~ low fM (5 orders of magnitude higher than traditional ELISA technique), with a wide dynamic range spanning 6 orders of magnitude (femto molar to nanomolar concentrations). PUBLIC HEALTH RELEVANCE: For many diseases, effective diagnosis and prognosis depends on the ability to quantitatively measure protein biomarkers which are present at low concentrations in clinical samples. Although blood serum is a rich source of such information, the challenge arises from the fact that it contains thousands of proteins whose concentrations range over 12 orders of magnitude (micromolar to femtomolar), making the analysis of rare protein markers extremely difficult. As a solution to this important problem, we propose to combine chip-based, high-gradient micro-magnetic separation technology with aptamer- based quantitative PCR to achieve quantitative, multiplexed protein detection with femtomolar detection sensitivities directly from undiluted serum samples.